Autophagy, a self-eating process, has attracted attention as a new target for anti-cell death engineering of recombinant Chinese hamster ovary (rCHO) cells in order to improve culture performance. In an effort to obtain genetic targets for autophagy control, changes in the mRNA and protein expression of four core autophagy pathway genes (Ulk1, Beclin-1, Atg7, and Atg9A) have been investigated in serum-free suspension cultures of rCHO cell lines. Among the four target genes, Atg9A was the only gene showing decreased levels of mRNA and protein simultaneously when comparing the expression in the late period of the culture showing the maximum level of autophagy with that in the control time point (Day 4). In order to verify the potential of Atg9A as an autophagy induction target, Atg9A was overexpressed in rCHO cells using the Tet-Off System. However, Atg9A overexpression did not significantly influence the autophagy induction and culture longevity. Taken together, the results obtained here demonstrate that the downregulation of Atg9A is not the sole limiting factor for autophagy induction in serum-free suspension cultures of rCHO cells. This suggests that combinatorial regulation of the genes in the upstream autophagy pathway with Atg9A overexpression could be a promising approach for autophagy control.

► The expression levels of core autophagy-related genes of rCHO cell lines were evaluated. ► The mRNA/protein level of Atg9A decreased in a later phase of culture. ► The controlled overexpression of Atg9A did not induce autophagy well. ► The combinatorial regulation could be a promising approach for autophagy induction.